The lack of improvement in cure rate of many common neoplasms is amply documented and often ascribed to failure of early detection. Present clinical means for detecting neoplastic tissue remain in many instances a gross anatomic procedure relying upon various physical findings or radiographic imaging procedures to select a site for histologic sampling. Scintillation imaging techniques with radiopharmaceuticals such as .sup.67 Ga-Gallium citrate, .sup.111 In-Bleomycin and .sup.131 I-Diiodofluorescein have limited success. These radiolabeled compounds lack specificity and sensitivity, that is, they are not preferentially taken up by neoplasms or tumors. Both .sup.67 Ga-Gallium citrate and .sup.111 In-Bleomycin are accumulated in inflammatory or infectious lesions. Currently, all available diagnostic techniques have many drawbacks and limitations in addition to lack of sensitivity and specificity. These include the use of traumatic invasive procedures and potential for serious complications.
Attempts to "mark" or "tag" cancer cells in order to differentiate them from normal tissues are not new. Various fluorescent compounds such as porphyrins, tetracycline derivatives, acridine orange and toluidine blue or radioactive isotopes have been extensively investigated. With the exception of porphyrin compounds, none of these substances used by earlier investigators are capable of routinely identifying and delineating malignant lesions.
To be effective, an ideal marker substances should: (1) be safe and non-toxic in humans; (2) selectively accumulate only in neoplastic tissue and not be taken up by normal or inflammatory tissues; (3) be simple to use and involve non-invasive procedures; (4) be capable of being documented by photographs, radiographs or other recording devices. The ideal marker or tracer continues to remain elusive, but derivatives of porphyrin appear to satisfy several of these requirements.
Porphyrins are complex tetrapyrrole compounds normally found in plants and in animals. They perform many vital biological functions by combining with metallic ions such as iron, magnesium, manganese, zinc, etc . . . to form metalloporphyrins. Metalloporphyrins such as hemoglobin, myosin, vitamin B-12, cytochrome, catalase, peroxidase and chlorophyll are essential for the normal metabolism of plants and animals. Many of these porphyrins and metalloporphyrins exhibit strong fluorescence when exposed to an appropriate exciting light source.
Hematoporphyrin, an artificial porphyrin compound, is prepared by treating hemoglobin with concentrated sulfuric acid (H.sub.2 SO.sub.4). It is a crude mixture of several porphyrins. The exact chemical composition of hematoporphyrin has not been identified.
The preferential affinity of porphyrins and hematoporphyrin for neoplastic tissue has been known for more than four decades. When injected into tumor-bearing animals, a brilliant red-orange fluorescence is produced by ultra violet(UV) light activation of the porphyrins or hematoporphyrin accumulated in the tumors. Hematoporphyrin appears to be a better tumor localizer than any porphyrin compounds.
Hematoporphyrin derivative (HPD), a recrystallized form of hematoporphyrin developed by Lipson and associates (Lipson, et al., J. Nat. Cancer Inst. 26: 1-11,1961) possesses higher tumor selectivity than earlier porphyrin compounds. It is currently the most actively investigated version of porphyrins for tumor identification and treatment. Clinical applications of HPD in tumor detection have been reported in the literature (Sanderson, et al., Cancer 30: 1368, 1972 and Kinsey, et al., Mayo Clin. Proc. 53: 594, 1978).
Despite the initial optimism over possible diagnostic application of HPD, the usefulness of this compound is limited. This is primarily due to the fact that HPD-fluorescence method involves invasive procedures. The fluorescence emitted by HPD must be activated in situ by a strong ultra violet light source which requires highly sophisticated endoscopic optical equipments. Visual observation of the tissue fluorescence at best is subjective & varies widely from different investigators. Quenching of the fluorescence by normal tissue, body fluids and blood is a major obstacle in achieving significant reliability and reproducibility of this technique. Endoscopic procedures often produce tissue damages which lead to hemmorhage and subsequent masking of the tumor. Another major problem is the inability to document photographically the fluorescence observed endoscopically. Complete reliance has to be placed on the visual interpretation and judgement of the endoscopist.
The use of radiolabeled HPD will eliminate most of the major problems encountered by the fluorescence-endoscopic method. Nuclear medicine procedures employing radiopharmaceuticals are simple and non-invasive. Following parenteral administration of the radiolabeled HPD, the radioactivity which concentrated in the neoplastic lesions can be easily detected and documented by scintigraphic imaging techniques.
Various porphyrin compounds had been labeled with radionuclides such as .sup.64 Cu and .sup.57 Co. Protoporphyrin and hematoporphyrin labeled with .sup.64 Cu were shown to concentrated in mouse tumors but failed to achieve significant tumor uptake in human beings (Base, R. et al, Cancer 11: 259, 1958). Similar results were obtained with .sup.57 Co-labeled hemtoporphyrin (Anghileri, LJ, et al: Nucl. Med. 15: 183, 1976). Earlier failures to localize human neoplasms with radiolabeled porphyrin compounds were attributed to: (1) poor labeling methodology; (2) the radionuclides used in the labeling process were incompatible with conventional scintigraphic imaging equipment; (3) in vivo instability of the labeled porphyrins; (4) alteration in biochemical properties after labeling process.
Technetium-99m (.sup.99m Tc) based radiopharmaceuticals have been widely used in the past 15 years. They are by far the safest and the most useful scintigraphic imaging agents developed for Nuclear Medicine procedures. The radionuclide .sup.99m Tc has many advantages. It is a pure gamma emitter with a relatively short physical half life of six hours. The gamma photon of 140 KeV energy is compatible with existing conventional scintillation imaging equipments. .sup.99m Tc-radiopharmaceuticals can be administered to patients in a much larger dose than many other radiolabeled compounds but produces a minimal radiation health hazard.
Currently, all .sup.99m Tc-based radiopharmaceuticals are produced based on the stannous(Sn)-acid reduction method (Eckelman, W. C., U.S. Pat. No. 3,725,295, 4/73). According to the labeling methodology, .sup.99m Tc(+7) in the stable form of sodium pertechnetate (Na.sup.99m TcO.sub.4) is first reduced to a chemically active (+4) or (+5) valence state with a reducing agent such as tin chloride(stannous chloride, SnCl.sub.2) which is dissolved in weak hydrochloric acid (HCl). Reduction of .sup.99m Tc-pertechnetate occurs at acidic condition with a pH of less than 2. An aqueous solution of the compound to be labeled is added to the reduced .sup.99m Tc/Sn(II) acidic mixture with the subsequent covalent binding of the radionuclide to the compound. The final mixture is then adjusted to pH 4-6 with a suitable buffer. The exact labeling mechanism is not known. With the exception of the chelates, other compounds or biological substances such as human serum albumin and red blood cells labeled by the Sn(II)-acid reduction method are completely denatured with significant loss of biochemical and physiological properties.
An alkaline Sn(II)-reduction method of labeling protein substances with .sup.99m Tc has been reported (Abramovici, et al, U.S. Pat. No. 4,057,617, 11/77). According to this invention, the proteins antibody and fibrinogen have claimed to be labeled with .sup.99m Tc at pH 11.6 condition. Technetium-99m pertechnetate is first reduced at pH 11.6 by adding an alkaline solution containing stannous chloride, acetic acid and sodium hydroxide(NaOH). The protein solution is then brought in contact with the reduced .sup.99m Tc forming a radiolabeled protein. The mixture is readjusted to pH 7.4 with a suitable buffer. The problems encountered by labeling protein substances at alkaline pH condition are similar to the acid reduction method, namely; protein denaturation, formation of insoluble radioactive tin colloids, protein degradation products, free or unbound .sup.99m Tc and very low yield. The labeling process of Abramovici is ineffective for producing useful radioactive tracer materials.
A simple chemical method of labeling protein substances with .sup.99m Tc under physiological condition has been developed by the present inventor (Wong, D. W., U.S. Pat. No. 4,293,537, 10/81 and Wong, D. W., et al, Int. J. Appl. Rad. Isotopes 29: 251, 1978). The basic labeling process involves the production of a chemically active .sup.99m Tc-(Sn)citrate complex species at pH 7.4 condition following initial reduction of .sup.99m Tc-pertechnetate. An aqueous protein solution is brought in contact with the radioactive complexing species forming a stable radiolabeled product. Unlike existing labeling processes, the actual labeling of the protein ligand with .sup.99m Tc occurs at physiological condition. Experimental data have confirmed that plasma proteins such as fibrinogen, antibodies, protein enzymes and hormones labeled with .sup.99m Tc by the physiologic chemical process are not denatured but retain their natural biochemical and immunological properties (Wong, D. W. et al, J. Nucl. Med. 20: 967, 1979 1979 and Wong, D. W. et al, J. Nucl. Med. 22: 229, 1981). Further investigation of this labeling process indicates that the .sup.99m Tc-(Sn)citrate complex species can tag many other compounds such as chelates and porphyrins in addition to protein substances.
Technetium-99m labeled hematoporphyrin derivative (.sup.99m Tc-HPD) offers many advantages over the fluorescence-endoscopic technique. Among these are: (1) the radionuclide .sup.99m Tc is firmly bound to the HPD ligand; (2) .sup.99m Tc-HPD is biologically active and remains stable in vivo; (3) it exhibits strong fluorescence when activated by UV light source; (4) Unlike other radiolabeled metalloporphyrins, .sup.99m Tc-HPD is preferentially accumulated by neoplastic tissue which can be documented by scintigraphic imaging techniques; (5) it can be administered to patients by parenteral routes; (6) it is safe and non-toxic; (7) the labeling process is so simple that a non-radioactive labeling reagent kit with long shelve life can be prepared in advance prior to actual use.